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Aptamer-Functionalized Barcode Particles for the Capture and Detection of Multiple Types of Circulating Tumor Cells Fuyin Zheng, Yao Cheng, Jie Wang, Jie Lu, Bin Zhang, Yuanjin Zhao,* and Zhongze Gu* The metastasis of cancer throughout the body is driven by circulating tumor cells (CTCs).[1–4] These cells are shed from the primary tumor into the bloodstream, travel to different tissues, and act as the cellular origin of cancer metastases. Aside from the conventional diagnostic approaches of tumor biopsy,[5,6] imaging,[7,8] and serum marker detection,[9,10] the detection of CTCs in peripheral blood provides valuable tumor information. CTCs have an extremely low abundance, comprising as few as one in 109 hematologic cells; hence their isolation and detection presents a tremendous technical challenge.[11–13] Existing microstructures,[14,15] trapping arrays,[16] or microfilterincorporated microfluidic[17–19] technologies have improved the recovery of CTCs from cancer patients; however, these technologies usually perform the simple function of cell capture or release. They cannot effectively distinguish between different CTCs, and thus they lack the functionality that would be the key for the diagnosis of specific cancers. Therefore, the development of a new platform that can capture, detect, and release multiple types of CTCs would dramatically increase the use of CTCs in diagnostics and prognostics. Barcode particles, whose encoding information enable simple identification, are attracting a great deal of interest in the field of multiple bioassays.[20–25] Many encoding strategies have been proposed for the barcode particles, including electronic, graphical, and spectrometric encoding.[26–30] However, traditional barcode particles typically have two unfavorable characteristics; they are small—on the order of micrometers— making them unsuitable as a substrate for cell capture and cell culturing, or when their surfaces would be covered by cells, their encoding information would be obscured or incomprehensible.[31–33] These characteristics, together with the debatable sensitivity, reliability, and specificity of the general surface morphology and biochemical modification, have limited the application of barcode particles in the capture and detection of multiple types of cells. Thus, the development of new barcode particles with distinct advantages is still required. In this paper, we present a new type of barcode particle with the desired capabilities; it has the ability to capture, detect, Dr. F. Y. Zheng,[+] Dr. Y. Cheng,[+] Dr. J. Wang, Dr. J. Lu, B. Zhang, Prof. Y. J. Zhao, Prof. Z. Z. Gu State Key Laboratory of Bioelectronics Southeast University Nanjing 210096, China E-mail: [email protected]; [email protected] Prof. Y. J. Zhao, Prof. Z. Z. Gu Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou Suzhou 215123, China [+]These authors contributed equally to this work.
DOI: 10.1002/adma.201403530
Adv. Mater. 2014, DOI: 10.1002/adma.201403530
and release multiple types of CTCs. The particles are spherical colloidal crystal clusters decorated with dendrimer-amplified aptamer probes. Their size can be adjusted to suit cell dimensions by their microfluidic-droplet templates. Encoding information in the particles is made possible by the characteristic reflection peaks originating from the photonic bandgap (PBG) structure of the colloidal crystals, and thus they are very stable. The encoding remains constant during multiple events of cell capture and cell culturing at the surface. In addition, because the spherical surface of the barcode particles has ordered nanoparticles of hexagonal symmetry, the substrates can not only provide more surface area for probe immobilization and reaction, but they can also provide a nanopatterned platform for highly efficient bioreactions. Moreover, by decorating the barcode particles with highly branched dendrimer-amplified aptamer probes, the sensitivity, reliability, and specificity of the cell capture are significantly increased. It was also demonstrated that the captured CTCs could be sorted accurately and controllably released with high viability. These features make the barcode particles ideal for capturing, detecting, and releasing multiple types of CTCs from clinical samples during cancer diagnostics and prognostics. In a typical experiment, the barcode particles were fabricated by the evaporation of droplet templates containing monodisperse silica nanoparticles. During this process, the nanoparticles form spherical assemblies with a regular arrangement (Figure 1a), and when the droplet were dried, the barcode particles were obtained. The nanoparticles of the barcode particles mainly formed a close-packed colloidal crystal array structure in the sphere. The PBGs and characteristic reflection peaks are a result of this structure. The peak position (λ) is determined by the structural period (d) and the average refractive index (neff) of the dielectric system according to the Bragg–Snell equation, λ = 2d(neff2–cos2θ)1/2, where θ is the Bragg angle of incidence of the light falling on the nanostructures. Therefore, by varying the diameter of the nanoparticles (and thus, the structural period d), a large number of the barcode particles with different diffraction peak positions for encoding could be obtained. However, to avoid code interference, only about 40 types of barcode particles can be used simultaneously in the visible-light wavelength range; this is generally enough for the demand of clinical diagnoses. Seven barcode particles with distinct colors are presented in Figure 1b. Because the barcode particles were derived from microfluidic droplets that contained silica nanoparticles, their size could be customized from several to hundreds of micrometers, not only by varying the flow rates of the water and oil phases, but also by using different concentrations of the silica nanoparticles for droplet generation. Here, barcode particles with a diameter of about 270 µm were generated for the cell-capture functionality.
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Figure 1. a) Scanning electron microscopy (SEM) images of the barcode particles (left) and of the enlarged nanostructure surface of the barcode particles, revealing the nanoparticle array (right). Scale bars are 200 µm (left figure) and 1 µm (right figure). b) Microscopy images (insets) and reflection spectra of seven different types of barcode particles. c) Schematic of the barcode particles used for enhanced CTC capture; the surface of the barcode particles is decorated with dendrimer and DNA aptamers.
The process of CTC capture on the surface of the barcode particles is illustrated in Figure 1c. Generally, the surface chemistry and topography of the substrates had a significant effect on the cell capture and culturing that occurred at the interface between the substrate materials and cells. In our study, the achieved barcode particles were assembled by silica nanoparticles, and thus they not only provided a large surface area for probe immobilization and reaction, but they also provided a close-packed hemispheric array surface topography for cell capture. Because of the reduction in the steric hindrance of the molecules via the artificial separation imposed by the nanopattern, the probes on this platform were much freer to react with the specific complementary parts of the cells, and the efficiency of cell capture increased. However, because of the small number of chemical groups on the silica surface that enable probe immobilization and cell adhesion, the practical capture density of cells on the barcode particles is limited. In addition, the effect of the nanopatterned surface topography on cell capture is still in need of improvement. To solve these problems, we etched the barcode particles to form a cell-preferred non-close-packed spherical array surface topography for CTC-capture functionality. We decorated the particles with highly branched dendrimer-amplified aptamer probes to improve the sensitivity, reliability, and specificity of capture and detection. It was found that the non-close-packed spherical array surface topography could provide a more effective platform for interactions with the cellular microvilli or filopodia (Figure 2a,b). This result was ascribed to the synergistic effects of the improved nanopatterned surface topography and the increased probe density. The latter was confirmed by
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the fluorescent-labeled hybridization reaction with the probes, which indicated that the probe density on the non-close-packed spherical array surface was about three times that on the closepacked spherical array surface (image available in Supporting Information (SI): Figure S1). When using the generation-5 (G5) poly(amidoamine) (PAMAM) dendrimer to decorate the barcode particles and immobilize the probe, the density of the probe was about one order of magnitude higher than without G5 PAMAM.[34–39] Under these conditions, the interaction of the barcode particles and the cells was very evident, and the corresponding capture efficiency was enhanced (Figure 2c). These improvements of the cell-capture functionality of these differently treated barcode particles were also confirmed by confocal microscopy and by the statistics of the distribution of cells on the surface of the particles (SI: Figure S2). Hence, the etched barcode particles with the dendrimer-amplified probes were used for further investigation of cell capture. The probes used in our work were cell-affinitive DNA aptamers, which could be quickly and reproducibly synthesized in an iterative process to specifically amplify sequences in vitro. They were single-stranded oligonucleotides having high and specific binding affinity to a target molecule on the cancer cells. In comparison with protein antibodies (e.g., anti-epithelial cell-adhesion molecule), which could interact with many cancer cells, the DNA aptamers could sort multiple types of cancer cells because of their high specificity to a certain cancer cell type. In addition, the aptamers had some inherent advantages for cell research, such as long-term stability, sustained reversible denaturation, nontoxicity, low immunogenicity, and short blood residence time. Therefore,
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conditions, the capture efficiency of the barcode particles could reach 90%. An important requirement for using barcode particles for multiple-CTC capture and detection is the accurate identification of their coded information during the entire process of cell capture. In general, encoding strategies, such as fluorescent dyes, quantum dots, and photopatterning, can achieve a large number of distinct barcodes. However, when cells are captured on the surface of these barcode particles, the barcodes might either become difficult to read because of the coverage of the cell layer or be affected by cell staining because of optical interference. Both of these would cause a reading error when extracting the encoded information of barcode particles, and thus result in incorrect multiplexing results. Our barcode particles overcame this problem by using their characteristic reflection peaks as their encoded elements. When the CTCs were captured Figure 2. Field-emission SEM (FESEM) images of the surface nanostructure (left row), of the and cultured on the barcode particles, they morphology of a captured CTC (middle row), and of the distribution of the CTCs (right row) of only interacted with the surface probes and various barcode-particle substrates: a) untreated barcode particles decorated with aptamers, did not affect the periodic structure of the b) etched barcode particles decorated with aptamers, c) etched barcode particles decorated whole microsphere. Thus, the encoded elewith dendrimers and aptamers. ments of our barcode particles, which were based on the physical structure of the materials, remained constant during cell adhesion (SI: Figure S5). the aptamers are considered to be ideal recognition elements Another advantage of our barcode particles for the cell-capture for the capture of target cancer cells. Here, three aptamers of and -detection functionalities was that only the fluorescent “TD05”, “Sgc8”, and “Sgd5” segments were synthesized and dyes for cell staining were used; dyes were not needed for the used. The first two aptamers demonstrated a specific affinity codes (SI: Figure S6). Thus, there was not any interference to Ramos and “CCRF-CEM” cells (SI: Figure S3); Ramos cells when cell fluorescence occurred (Figure 3), which avoided the are from a human lymphoma cell line while CCRF-CEM cells fluorescence spectral overlap problem that exists in most colororiginate from human leukemia. encoded particles. To achieve high CTC-capture efficiency on the aptamer-decorated barcode particles, the dendrimer concentration, aptamer To demonstrate the reliability of the barcode particles in concentration, capture time, and temperature were investigated capturing and detecting multiple types of CTCs, three types of (SI: Figure S4). When the concentration of the dendrimer was barcode particles with their characteristic reflection peak posigradually increased to 500 µg/mL, the density of captured tions at 661, 583, and 447 nm (exhibiting red, green, and blue cells on the surface of the barcode particles also increased—to structural colors) were modified with three types of aptamers— (66 583)±(4041) cells/cm2. The density gradually saturated when namely TD05, Sgc8, and Sgd5, respectively. These aptamermodified barcode particles were then mixed and incubated in a the concentration exceeded 500 µg/mL. The effect of aptamer multitarget solution of green (calcein AM)-stained Ramos cells concentration on the density of captured cells was similar to and blue (Hoechst 33342)-stained CCRF-CEM cells (Figure 4a). that of the dendrimer concentration. The density of the capBecause of the specific binding between the aptamers and their tured cells saturated when the aptamer concentration reached corresponding target CTCs, we expected to observe CTCs on 50 µM. Regarding the capture time, the number of captured the surface of the barcode particles when their corresponding cells initially increased with incubation time; it then reached aptamer probes were present. The results of the capture and a plateau at an incubation time of 40 min (SI: Figure S4c). detection of the cells are shown in Figure 4b–g. It was found This value indicated a much faster reaction and capture prothat the green cells were mainly enriched on the red barcode cess than the previous method—better by about 60 min—and particles, and the blue cells were mainly enriched on the green this could be ascribed to the intrinsic advantages of the higher barcode particles, whereas the control group of blue barcode flexibility and faster reaction kinetics of the barcode particles in particles showed no obvious cell capture. These results were solution because of the radial diffusion of the particles and the consistent with the content of the sample to which the barcode targets. For the capture temperature, the aptamer TD05 had a particles were exposed. Further analysis found that the capture slightly higher binding affinity for the Ramos cell at 4 °C than specificities for the Ramos and CCRF-CEM cells were 98 ± 1.3% at 37 °C (SI: Figure S4d). This might be because the conformaand 97 ± 1.8%, respectively, similar to the levels achieved with tion of the aptamer TD05 at 4 °C is more helpful in stabilizing microfluidic cell-affinity chromatography devices. After the the bonds with cell surface receptors. Under these optimized
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Figure 3. Laser scanning confocal microscopy (LSCM) images of captured CTCs on different substrates: a) untreated barcode particles decorated with aptamers, b) etched barcode particles decorated with aptamers, c) etched barcode particles decorated with dendrimers and aptamers. From left to right: cross-section white-light images, cross-section fluorescent images, 3D fluorescent images, and merged 3D fluorescent and white-light images. Scale bar indicates 100 µm.
process of capture, the detection of different CTCs could also be carried out by reading the fluorescence signals of the cells on the surface of the barcode particles. For the cellular and molecular investigation, the captured CTCs could be released on demand from the barcode particles by using exonuclease I to degrade single-stranded DNA aptamer probes. The release efficiency of the CTCs from the barcode particles was about 86.6 ± 3.8% (SI: Figure S7a). The activity in the released CTCs was 97% (SI: Figure S7b). These data reveal the ability of our approach to release undamaged CTCs. Based on this feature, the barcode particles were employed in the investigation of CTCs in real clinical samples. After the capture of the various CTCs, the cells were treated with a post-staining method with fluorescently labeled specific antibodies: CD3-FITC targeting CCRF-CEM cells and CD19-APC targeting Ramos cells (FITC and APC are the fluorescent labels, fluorescein isothiocyanate and allophycocyanate, respectively). The CTCenriched barcode particles were then sorted
Figure 4. a) Scheme showing the barcode particles capturing multiple types of CTCs. Various aptamers, TD05, Sgc8, and Sgd5, were used; and greenand blue-stained cells were used as the target cells. b) Optical microscopy image, c) green fluorescent image, and d) blue fluorescent image of the barcode particles after the capture of the various CTCs. e–g) Enlarged images of b–d, respectively. Due to the overlap of the blue Hoechst 33342 and green calcein AM, the Hoechst 33342 were also excited appearing green in c and f.
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based on their encoded colors. Finally, the CTCs were released from the particles and analyzed by a flow cytometer. Using multiple-CTC capture, the amount of CTCs in the real clinical samples were decreased from 5.3% to 0.38% (Figure 5a,b), which indicated a high capture efficiency of 92.83%. Further analysis of the CTCs from the red and green barcode particles, respectively, indicated that 96.8% of the released cells from the red barcode particles were the CCRF-CEM cells, 98.94% of the released cells from the green barcode particles were the Ramos cells (Figure 5c,d). These results not only indicated the high specificity of the aptamer probes to their corresponding target CTCs, but it also demonstrated the high accuracy of the barcode particles for the multiple-CTC capture in the real clinical samples. In summary, we developed a novel barcode-particle technology that can simultaneously capture, detect, and release multiple types of CTCs from a complex sample. The barcode particles are spherical colloidal crystal clusters with their characteristic reflection peak as their code, which remains stable during cell capture and culturing on their surfaces. The surface of the barcode particles was etched to have a non-closepacked spherical array topography, which provided not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions.
Adv. Mater. 2014, DOI: 10.1002/adma.201403530
In addition, by decorating the barcode particles with highly branched dendrimer-amplified aptamer probes, the sensitivity, reliability, and specificity of the cell capture were significantly increased. These features make the barcode particles ideal for capturing, detecting, and subsequently releasing various CTCs. We anticipate that this technology may open up new horizons in clinical cancer diagnostics and prognostics.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was supported by the National Natural Science Foundation of China (Grants 21473029, 91227124, and 21327902), the National Science Foundation of Jiangsu (Grant No. BK20140028), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1222), and the Technology Invocation Team of Qinglan Project of Jiangsu Province.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: August 3, 2014 Published online:
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Aptamer-Functionalized Barcode Particles for the Capture and Detection of Multiple Types of Circulating Tumor Cells Fuyin Zheng, Yao Cheng, Jie Wang, Jie Lu, Bin Zhang, Yuanjin Zhao,* and Zhongze Gu* The metastasis of cancer throughout the body is driven by circulating tumor cells (CTCs).[1–4] These cells are shed from the primary tumor into the bloodstream, travel to different tissues, and act as the cellular origin of cancer metastases. Aside from the conventional diagnostic approaches of tumor biopsy,[5,6] imaging,[7,8] and serum marker detection,[9,10] the detection of CTCs in peripheral blood provides valuable tumor information. CTCs have an extremely low abundance, comprising as few as one in 109 hematologic cells; hence their isolation and detection presents a tremendous technical challenge.[11–13] Existing microstructures,[14,15] trapping arrays,[16] or microfilterincorporated microfluidic[17–19] technologies have improved the recovery of CTCs from cancer patients; however, these technologies usually perform the simple function of cell capture or release. They cannot effectively distinguish between different CTCs, and thus they lack the functionality that would be the key for the diagnosis of specific cancers. Therefore, the development of a new platform that can capture, detect, and release multiple types of CTCs would dramatically increase the use of CTCs in diagnostics and prognostics. Barcode particles, whose encoding information enable simple identification, are attracting a great deal of interest in the field of multiple bioassays.[20–25] Many encoding strategies have been proposed for the barcode particles, including electronic, graphical, and spectrometric encoding.[26–30] However, traditional barcode particles typically have two unfavorable characteristics; they are small—on the order of micrometers— making them unsuitable as a substrate for cell capture and cell culturing, or when their surfaces would be covered by cells, their encoding information would be obscured or incomprehensible.[31–33] These characteristics, together with the debatable sensitivity, reliability, and specificity of the general surface morphology and biochemical modification, have limited the application of barcode particles in the capture and detection of multiple types of cells. Thus, the development of new barcode particles with distinct advantages is still required. In this paper, we present a new type of barcode particle with the desired capabilities; it has the ability to capture, detect, Dr. F. Y. Zheng,[+] Dr. Y. Cheng,[+] Dr. J. Wang, Dr. J. Lu, B. Zhang, Prof. Y. J. Zhao, Prof. Z. Z. Gu State Key Laboratory of Bioelectronics Southeast University Nanjing 210096, China E-mail: [email protected]; [email protected] Prof. Y. J. Zhao, Prof. Z. Z. Gu Laboratory of Environment and Biosafety Research Institute of Southeast University in Suzhou Suzhou 215123, China [+]These authors contributed equally to this work.
DOI: 10.1002/adma.201403530
Adv. Mater. 2014, DOI: 10.1002/adma.201403530
and release multiple types of CTCs. The particles are spherical colloidal crystal clusters decorated with dendrimer-amplified aptamer probes. Their size can be adjusted to suit cell dimensions by their microfluidic-droplet templates. Encoding information in the particles is made possible by the characteristic reflection peaks originating from the photonic bandgap (PBG) structure of the colloidal crystals, and thus they are very stable. The encoding remains constant during multiple events of cell capture and cell culturing at the surface. In addition, because the spherical surface of the barcode particles has ordered nanoparticles of hexagonal symmetry, the substrates can not only provide more surface area for probe immobilization and reaction, but they can also provide a nanopatterned platform for highly efficient bioreactions. Moreover, by decorating the barcode particles with highly branched dendrimer-amplified aptamer probes, the sensitivity, reliability, and specificity of the cell capture are significantly increased. It was also demonstrated that the captured CTCs could be sorted accurately and controllably released with high viability. These features make the barcode particles ideal for capturing, detecting, and releasing multiple types of CTCs from clinical samples during cancer diagnostics and prognostics. In a typical experiment, the barcode particles were fabricated by the evaporation of droplet templates containing monodisperse silica nanoparticles. During this process, the nanoparticles form spherical assemblies with a regular arrangement (Figure 1a), and when the droplet were dried, the barcode particles were obtained. The nanoparticles of the barcode particles mainly formed a close-packed colloidal crystal array structure in the sphere. The PBGs and characteristic reflection peaks are a result of this structure. The peak position (λ) is determined by the structural period (d) and the average refractive index (neff) of the dielectric system according to the Bragg–Snell equation, λ = 2d(neff2–cos2θ)1/2, where θ is the Bragg angle of incidence of the light falling on the nanostructures. Therefore, by varying the diameter of the nanoparticles (and thus, the structural period d), a large number of the barcode particles with different diffraction peak positions for encoding could be obtained. However, to avoid code interference, only about 40 types of barcode particles can be used simultaneously in the visible-light wavelength range; this is generally enough for the demand of clinical diagnoses. Seven barcode particles with distinct colors are presented in Figure 1b. Because the barcode particles were derived from microfluidic droplets that contained silica nanoparticles, their size could be customized from several to hundreds of micrometers, not only by varying the flow rates of the water and oil phases, but also by using different concentrations of the silica nanoparticles for droplet generation. Here, barcode particles with a diameter of about 270 µm were generated for the cell-capture functionality.
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Figure 1. a) Scanning electron microscopy (SEM) images of the barcode particles (left) and of the enlarged nanostructure surface of the barcode particles, revealing the nanoparticle array (right). Scale bars are 200 µm (left figure) and 1 µm (right figure). b) Microscopy images (insets) and reflection spectra of seven different types of barcode particles. c) Schematic of the barcode particles used for enhanced CTC capture; the surface of the barcode particles is decorated with dendrimer and DNA aptamers.
The process of CTC capture on the surface of the barcode particles is illustrated in Figure 1c. Generally, the surface chemistry and topography of the substrates had a significant effect on the cell capture and culturing that occurred at the interface between the substrate materials and cells. In our study, the achieved barcode particles were assembled by silica nanoparticles, and thus they not only provided a large surface area for probe immobilization and reaction, but they also provided a close-packed hemispheric array surface topography for cell capture. Because of the reduction in the steric hindrance of the molecules via the artificial separation imposed by the nanopattern, the probes on this platform were much freer to react with the specific complementary parts of the cells, and the efficiency of cell capture increased. However, because of the small number of chemical groups on the silica surface that enable probe immobilization and cell adhesion, the practical capture density of cells on the barcode particles is limited. In addition, the effect of the nanopatterned surface topography on cell capture is still in need of improvement. To solve these problems, we etched the barcode particles to form a cell-preferred non-close-packed spherical array surface topography for CTC-capture functionality. We decorated the particles with highly branched dendrimer-amplified aptamer probes to improve the sensitivity, reliability, and specificity of capture and detection. It was found that the non-close-packed spherical array surface topography could provide a more effective platform for interactions with the cellular microvilli or filopodia (Figure 2a,b). This result was ascribed to the synergistic effects of the improved nanopatterned surface topography and the increased probe density. The latter was confirmed by
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the fluorescent-labeled hybridization reaction with the probes, which indicated that the probe density on the non-close-packed spherical array surface was about three times that on the closepacked spherical array surface (image available in Supporting Information (SI): Figure S1). When using the generation-5 (G5) poly(amidoamine) (PAMAM) dendrimer to decorate the barcode particles and immobilize the probe, the density of the probe was about one order of magnitude higher than without G5 PAMAM.[34–39] Under these conditions, the interaction of the barcode particles and the cells was very evident, and the corresponding capture efficiency was enhanced (Figure 2c). These improvements of the cell-capture functionality of these differently treated barcode particles were also confirmed by confocal microscopy and by the statistics of the distribution of cells on the surface of the particles (SI: Figure S2). Hence, the etched barcode particles with the dendrimer-amplified probes were used for further investigation of cell capture. The probes used in our work were cell-affinitive DNA aptamers, which could be quickly and reproducibly synthesized in an iterative process to specifically amplify sequences in vitro. They were single-stranded oligonucleotides having high and specific binding affinity to a target molecule on the cancer cells. In comparison with protein antibodies (e.g., anti-epithelial cell-adhesion molecule), which could interact with many cancer cells, the DNA aptamers could sort multiple types of cancer cells because of their high specificity to a certain cancer cell type. In addition, the aptamers had some inherent advantages for cell research, such as long-term stability, sustained reversible denaturation, nontoxicity, low immunogenicity, and short blood residence time. Therefore,
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conditions, the capture efficiency of the barcode particles could reach 90%. An important requirement for using barcode particles for multiple-CTC capture and detection is the accurate identification of their coded information during the entire process of cell capture. In general, encoding strategies, such as fluorescent dyes, quantum dots, and photopatterning, can achieve a large number of distinct barcodes. However, when cells are captured on the surface of these barcode particles, the barcodes might either become difficult to read because of the coverage of the cell layer or be affected by cell staining because of optical interference. Both of these would cause a reading error when extracting the encoded information of barcode particles, and thus result in incorrect multiplexing results. Our barcode particles overcame this problem by using their characteristic reflection peaks as their encoded elements. When the CTCs were captured Figure 2. Field-emission SEM (FESEM) images of the surface nanostructure (left row), of the and cultured on the barcode particles, they morphology of a captured CTC (middle row), and of the distribution of the CTCs (right row) of only interacted with the surface probes and various barcode-particle substrates: a) untreated barcode particles decorated with aptamers, did not affect the periodic structure of the b) etched barcode particles decorated with aptamers, c) etched barcode particles decorated whole microsphere. Thus, the encoded elewith dendrimers and aptamers. ments of our barcode particles, which were based on the physical structure of the materials, remained constant during cell adhesion (SI: Figure S5). the aptamers are considered to be ideal recognition elements Another advantage of our barcode particles for the cell-capture for the capture of target cancer cells. Here, three aptamers of and -detection functionalities was that only the fluorescent “TD05”, “Sgc8”, and “Sgd5” segments were synthesized and dyes for cell staining were used; dyes were not needed for the used. The first two aptamers demonstrated a specific affinity codes (SI: Figure S6). Thus, there was not any interference to Ramos and “CCRF-CEM” cells (SI: Figure S3); Ramos cells when cell fluorescence occurred (Figure 3), which avoided the are from a human lymphoma cell line while CCRF-CEM cells fluorescence spectral overlap problem that exists in most colororiginate from human leukemia. encoded particles. To achieve high CTC-capture efficiency on the aptamer-decorated barcode particles, the dendrimer concentration, aptamer To demonstrate the reliability of the barcode particles in concentration, capture time, and temperature were investigated capturing and detecting multiple types of CTCs, three types of (SI: Figure S4). When the concentration of the dendrimer was barcode particles with their characteristic reflection peak posigradually increased to 500 µg/mL, the density of captured tions at 661, 583, and 447 nm (exhibiting red, green, and blue cells on the surface of the barcode particles also increased—to structural colors) were modified with three types of aptamers— (66 583)±(4041) cells/cm2. The density gradually saturated when namely TD05, Sgc8, and Sgd5, respectively. These aptamermodified barcode particles were then mixed and incubated in a the concentration exceeded 500 µg/mL. The effect of aptamer multitarget solution of green (calcein AM)-stained Ramos cells concentration on the density of captured cells was similar to and blue (Hoechst 33342)-stained CCRF-CEM cells (Figure 4a). that of the dendrimer concentration. The density of the capBecause of the specific binding between the aptamers and their tured cells saturated when the aptamer concentration reached corresponding target CTCs, we expected to observe CTCs on 50 µM. Regarding the capture time, the number of captured the surface of the barcode particles when their corresponding cells initially increased with incubation time; it then reached aptamer probes were present. The results of the capture and a plateau at an incubation time of 40 min (SI: Figure S4c). detection of the cells are shown in Figure 4b–g. It was found This value indicated a much faster reaction and capture prothat the green cells were mainly enriched on the red barcode cess than the previous method—better by about 60 min—and particles, and the blue cells were mainly enriched on the green this could be ascribed to the intrinsic advantages of the higher barcode particles, whereas the control group of blue barcode flexibility and faster reaction kinetics of the barcode particles in particles showed no obvious cell capture. These results were solution because of the radial diffusion of the particles and the consistent with the content of the sample to which the barcode targets. For the capture temperature, the aptamer TD05 had a particles were exposed. Further analysis found that the capture slightly higher binding affinity for the Ramos cell at 4 °C than specificities for the Ramos and CCRF-CEM cells were 98 ± 1.3% at 37 °C (SI: Figure S4d). This might be because the conformaand 97 ± 1.8%, respectively, similar to the levels achieved with tion of the aptamer TD05 at 4 °C is more helpful in stabilizing microfluidic cell-affinity chromatography devices. After the the bonds with cell surface receptors. Under these optimized
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Figure 3. Laser scanning confocal microscopy (LSCM) images of captured CTCs on different substrates: a) untreated barcode particles decorated with aptamers, b) etched barcode particles decorated with aptamers, c) etched barcode particles decorated with dendrimers and aptamers. From left to right: cross-section white-light images, cross-section fluorescent images, 3D fluorescent images, and merged 3D fluorescent and white-light images. Scale bar indicates 100 µm.
process of capture, the detection of different CTCs could also be carried out by reading the fluorescence signals of the cells on the surface of the barcode particles. For the cellular and molecular investigation, the captured CTCs could be released on demand from the barcode particles by using exonuclease I to degrade single-stranded DNA aptamer probes. The release efficiency of the CTCs from the barcode particles was about 86.6 ± 3.8% (SI: Figure S7a). The activity in the released CTCs was 97% (SI: Figure S7b). These data reveal the ability of our approach to release undamaged CTCs. Based on this feature, the barcode particles were employed in the investigation of CTCs in real clinical samples. After the capture of the various CTCs, the cells were treated with a post-staining method with fluorescently labeled specific antibodies: CD3-FITC targeting CCRF-CEM cells and CD19-APC targeting Ramos cells (FITC and APC are the fluorescent labels, fluorescein isothiocyanate and allophycocyanate, respectively). The CTCenriched barcode particles were then sorted
Figure 4. a) Scheme showing the barcode particles capturing multiple types of CTCs. Various aptamers, TD05, Sgc8, and Sgd5, were used; and greenand blue-stained cells were used as the target cells. b) Optical microscopy image, c) green fluorescent image, and d) blue fluorescent image of the barcode particles after the capture of the various CTCs. e–g) Enlarged images of b–d, respectively. Due to the overlap of the blue Hoechst 33342 and green calcein AM, the Hoechst 33342 were also excited appearing green in c and f.
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COMMUNICATION Figure 5. a,b) Flow cytometry scatter plots summarize the CTC and white blood cell (WBC) distribution before (a) and after (b) the cell-capture process. WBCs were bound with the fluorescent antibody CD45 PE/Cy7 which is associated with the gate FL3-A:FL3-A. c) Flow chart of the cell purities of the CCRF-CEM released from the green barcode particles. d) Flow chart of the cell purities of the Ramos cells released from the red barcode particles. CD3 FITC and CD19 APC were associated with the FL1-A:FL1-A and FL4-A:FL4-A gates, respectively.
based on their encoded colors. Finally, the CTCs were released from the particles and analyzed by a flow cytometer. Using multiple-CTC capture, the amount of CTCs in the real clinical samples were decreased from 5.3% to 0.38% (Figure 5a,b), which indicated a high capture efficiency of 92.83%. Further analysis of the CTCs from the red and green barcode particles, respectively, indicated that 96.8% of the released cells from the red barcode particles were the CCRF-CEM cells, 98.94% of the released cells from the green barcode particles were the Ramos cells (Figure 5c,d). These results not only indicated the high specificity of the aptamer probes to their corresponding target CTCs, but it also demonstrated the high accuracy of the barcode particles for the multiple-CTC capture in the real clinical samples. In summary, we developed a novel barcode-particle technology that can simultaneously capture, detect, and release multiple types of CTCs from a complex sample. The barcode particles are spherical colloidal crystal clusters with their characteristic reflection peak as their code, which remains stable during cell capture and culturing on their surfaces. The surface of the barcode particles was etched to have a non-closepacked spherical array topography, which provided not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions.
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In addition, by decorating the barcode particles with highly branched dendrimer-amplified aptamer probes, the sensitivity, reliability, and specificity of the cell capture were significantly increased. These features make the barcode particles ideal for capturing, detecting, and subsequently releasing various CTCs. We anticipate that this technology may open up new horizons in clinical cancer diagnostics and prognostics.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was supported by the National Natural Science Foundation of China (Grants 21473029, 91227124, and 21327902), the National Science Foundation of Jiangsu (Grant No. BK20140028), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1222), and the Technology Invocation Team of Qinglan Project of Jiangsu Province.
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Received: August 3, 2014 Published online:
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